GVH Direction Research Articles

Donor Natural Killer Cell Allorecognition of Missing Self in Haploidentical Hematopoietic Transplantation for Acute Myeloid Leukemia: Challenging Its Predictive Value.

In haploidentical transplants that are KIR ligand mismatched in the GvH direction, functional donor NK-cells that express as their sole inhibitory receptor for self, a KIR for the HLA-class-I group which is absent in the recipient, sense the missing expression of the self class-I ligand on allogeneic targets and mediate alloreactions. In a limited series of transplants donor-vs-recipient NK-cell alloreactivity reduced the risk of AML relapse and markedly improved EFS (Ruggeri L, Science 2002). Here, we analyzed 112 AML patients transplanted from NK-alloreactive (n=51) or non-NK-alloreactive (n=61) haploidentical donors. NK-alloreactive donors possessed: HLA-class-I KIR ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK-clones against recipient targets. Transplantation from NK-alloreactive donors was associated with: significantly lower relapse rate in patients transplanted in CR (3% vs 47%) (P<0.003); decrease infectious mortality which was more evident in patients transplanted in relapse (P=0.1); better EFS in patients transplanted in relapse (34% vs 6%, P=0.04) and in remission (67% vs 18%, P=0.02); reduced risk of relapse or death (relative risk vs non-NK-alloreactive donor: 0.48 [95% CI 0.29–0.78], P<0.001). Recently, an additional algorithm, termed the “missing ligand” model, has been proposed for predicting favorable outcomes not only in haploidentical (Leung W, JI 2004; JI 2005) but also in matched sibling (Hsu KC, Blood 2005) and in unrelated donor transplants (Hsu KC, BBMT 2006). The “missing-ligand” model includes all donor-recipient pairs in whom there is a mismatch between KIR(s) in the donor and HLA molecule(s) in the recipient. Consequently, the model includes all KIR ligand-mismatched transplants because they are all associated with a missing KIR ligand in the recipient, and KIR ligand-matched transplants from donors possessing “extra” KIR(s) for which neither donor nor recipient have HLA ligand(s). Therefore, in the same series of patients we tested the “missing ligand” model. The first step was to divide our 61 non-NK alloreactive (KIR ligand-matched) donor-recipient pairs according to the number of KIR ligands in donor and recipient, i.e., three KIR ligands (29 patients; 15 in remission, 14 in relapse at transplant) vs fewer than three (32 patients; 16 in remission, 16 in relapse at transplant). EFS did not differ in each sub-group. Both curves indicated worse survival than after transplantation from NK-alloreactive donors. The second step was to group the above 32 “missing ligand” transplants and all 51 KIR ligand-mismatched transplants (which corresponded to all our NK-alloreactive transplants). We analyzed EFS in this pool of 83 patients (46 in remission, 37 in relapse at transplant) against EFS in 29 patients with no missing ligand (15 in remission, 14 in relapse at transplant). No significant difference emerged. EFS in the “missing ligand” cohort was worse than after transplantation from NK-alloreactive donors. Therefore, the present analysis leaves no doubt that KIR ligand mismatches, i.e., donor NK cell recognition of “missing self” on recipient targets, are essential for triggering powerful NK cell alloreactions that impact beneficially on transplantation outcomes.

Toll-Like Receptors 2 and 4 Are Involved in the Induction of Graft-Versus-Host-Disease in Mice.

BACKGROUND: Intestinal Graft-versus-Host disease is a frequent and often lethal complication after allogenic stem cell transplantation. Since NOD2 polymorphisms have been recognized as potential triggers of severe intestinal GvHD in humans, we have developed murine transplantation models to investigate the role of different pattern recognition receptors (PRR) in GvHD and GvL. Here we report our results on the role of TLR2 and TLR4 for the induction of GvHD.METHODS: Severity of GvHD in wildtype (wt) C57B/10 (H-2Db), TLR2−/−, TLR4−/−, and combined TLR2−/−TLR4−/− C57B/10 mice was investigated. Mice received treosulfan 2000 mg/kg from day -3 to -1 and cyclophosphamide 200 mg/kg day -1 prior to injection of 10×10^6 H-2Dd BM cells and 5×10^6 splenocytes (SC). Survival and GvHD score were assessed daily. Engraftment was determined every 2 weeks in pB and at the end of the experiments in bone marrow by flow cytometry. T cell alloreactivity in GvH direction was assessed by MLR using splenocytes as stimulators from PRR-deficient mice or wt as control and CFSE-staining as read-out. The relevance of PRR ligands for the enhancement of GvH alloreactivity was determined by addition of lipid A, lipopetides, or CpG.RESULTS:in vivo data: The transfer of 10×10^6 BMC + 5×10^6 SC induced a severe GvHD in all wt recipients, leading to death of 90% of the animals within 20 days. Recipient mice lacking either TLR2 or TLR4 showed only a slightly and not significantly decreased GvHD lethality. In recipients lacking both PPRs, i.e. TLR2 and TLR4, GvHD was generally milder and the majority (60%) of the animals survived until day 20 (p<0.05). However, the long term survival was not significantly improved. Differences in clinical severity of GvHD were confirmed histologically.In vitro data: Stimulation with cells from TLR2−/− and TLR4−/− mice resulted in a decreased alloreactivity in MLR. A median of 2% of Balb/c CD4+ T cells proliferated in response to C57B/10 stimulators. The addition of the TLR2 and TLR4 ligands lipopeptide, Lipid A and CpG significantly (p<0.05) increased the proliferation of CD4+ T cells in a specific manner more than twofold.CONCLUSION: Our in vivo and in vitro data consistantly show that bacterial components are involved in triggering GvH alloreactivity via different types of PPRs. Binding of bacterial substances to TLR2 and TLR4 leads to activation of the immune system and subsequent induction of GvHD. Our data provide an experimental basis for the development of strategies for modulation of the intestinal gut flora by selective gut decontamination and/or probiotic regimens to prevent GvHD in humans.

Unmanipulated HLA 2-3 antigen-mismatched (haploidentical) nonmyeloablative stem cell transplantation

To expand the donor pool, allogeneic stem cell transplantation (SCT) using HLA 2-3 antigen-mismatched (haploidentical) related donors has been studied. To date, there are a few reports describing nonmyeloablative stem cell transplantation (NST) from HLA-haploidentical donors. We recently showed that a nonmyeloablative regimen consisting of fludarabine, busulfan, and ATG was sufficiently immunosuppressive to achieve donor type engraftment in transplantation from HLA-haploidentical donors who had 1 antigen-mismatch in the GVH direction and 2-3 antigen-mismatches in the HVG direction (Leukemia, 2003).

Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation

In haplo-identical hematopoietic transplantation, donor vs. recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory killer cell Ig-like receptors (KIR) for self-HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. When faced with mismatched allogeneic targets, these NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. KIR ligand mismatches in the GvH direction trigger donor vs. recipient NK cell alloreactions, which improve engraftment, do not cause GvHD and control relapse in AML patients . The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated in mouse models. The infusion of alloreactive NK cells ablates (i) leukemic cells, (ii) recipient T cells that reject the graft and (iii) recipient DC that trigger GvHD, thus protecting from GvHD.

Negative Impact of KIR-Ligand Mismatch on Transplant-Related Mortality (TRM) in Umbilical Cord Blood Transplant (UCBT) Recipients.

Umbilical cord blood (UCB) is frequently considered as a suitable alternate source of hematopoietic stem cells (HSC) for both pediatric and adult patients who require HSC transplant for treatment of high-risk or relapsed hematologic malignancy. As mismatched killer Ig-like receptor ligands (KIR-L MM) has been associated with anti-host alloreactive natural killer (NK) cell activity and reduced risk of acute myeloid leukemia relapse in recipients of T-cell depleted haploidentical HSC, we hypothesized that GVL after UCBT may also be mediated by NK alloreactivity. We therefore assessed the effect of KIR-L MM in 243 recipients of UCB transplanted at the University of Minnesota between 1998 and 2004 for whom HLA-A, -B, -C and DRB1 typing was available for both patient and UCB unit(s). Median age, weight, and follow-up were 27 yrs (range, 0.2–69), 64.6 kg (range, 3.8–120.2), and 1.1 yr (range, 0.5–6.6), respectively. For recipients of double UCBT (n =106), we analyzed the KIR-L assignment of the engrafting unit only. KIR-L MM in the GVH direction was established using the algorithm of Ruggeri and Velardi and was found in 70 (29%) donor-recipient pairs. Probability of 2-year survival was 54% (95%CI: 42–66) vs. 47% (95%CI: 38–56) (p=0.88) in KIR-L MM vs. matched pairs, respectively. Intensity of the conditioning regimen was the only independent predictor of survival, with a RR of death 1.54 (95%CI: 1.07–2.22, p=0.02) for those who received a non-myeloablative conditioning. For the whole group, incidence of relapse at 1-yr was 17% (95%CI: 8–26) vs. 31% (95%CI: 24–38) (p=0.12); for myeloid malignancy patients, incidence of relapse at 1 year was 13% (95%CI:2–24) vs. 34% (95%CI:22–46)(p=0.10). Intensity of the conditioning regimen was the only independent predictor of relapse, with a RR 2.09 (95%CI: 1.31–3.35, p<0.01) for those who received a non-myeloablative conditioning. Incidence of graft failure and grade II–IV acute GVHD was 11% (95%CI: 3–18) vs. 10% (95%CI: 6–15) (p= 0.97) and 50% (95%CI: 37–63) vs. 50% (95%CI: 42–58) (p=0.67), respectively. Notably, the incidence of TRM was higher among recipients of KIR-L MM grafts [26% (95%CI: 15–26) vs. 13% (95%CI: 8–18), p=0.01]. KIR-L MM did not impact on outcome within the subgroups of patients based on conditioning, number UCB units (single vs. double), and those who had a myeloid malignancy (AML, CML or MDS). In multivariate analysis, a nucleated cell dose < 3.5 X 10E7/kg [RR 3.86 (95%CI: 1.84–8.12, p<0.01)] and KIR-L MM [RR 2.15 (95%CI: 1.15–4.01, p<0.01)] were independent predictors of increased TRM. In summary, in the setting of UCBT, KIR-L MM is associated with increased TRM with no obvious beneficial effect on engraftment, relapse risk or survival as previously demonstrated in recipients of TCD haploidentical HSC. Differences in NK cells reconstitution, presence of T-cells and/or use of immunosuppression may interfere with any potential beneficial effect alloreactive NK cells in the setting of UCBT. While additional studies are still needed, results to date fail to support a specific search for UCB units with a KIR-L MM.

Minimal HLA Disparity and KIR Ligand Compatibility in Host Versus Graft Direction May Facilitate Donor Engraftment Following In Vivo and Ex Vivo T Cell Depleted (TCD) Nonmyeloablative Haploidentical Stem Cell Transplantation for Hematologic Malignancies.

Mixed chimerism (MC) can be induced in mice across MHC barriers using nonmyeloablative conditioning and T cell depletion. Delayed donor lymphocyte infusions (DLI) convert this MC state to full donor hematopoiesis and impart a powerful graft versus tumor effect without inducing GVHD. Based on this principle, we developed a clinical trial with a goal of inducing a GVH-free mixed chimeric platform for delayed cellular immunotherapy via DLI following nonmyeloablative haploidentical HSCT. This study analyzed graft outcomes in 13 patients who received in vivo and ex vivo TCD, haploidentical (HLA 1–3 Ag mismatched) HSCT for refractory hematologic malignancies (NHL, n=7; CLL, n=1; HD, n=3; CML, n=1; MDS, n=1). Conditioning consisted of MEDI-507 (siplizumab, an anti-CD2 mAb) for in vivo T cell depletion of both the host and donor, cyclophosphamide, fludarabine, thymic irradiation, and a brief course of cyclosporine. An earlier cohort of 4 patients did not receive fludarabine. All patients received ex vivo TCD of G-CSF-mobilized peripheral blood stem cells using a CD34+ cell-selection device. A median of 7.38 x 106 (3.19 x 106 to 1.49 x 107) CD 34+ cells/kg and a median of 5.24 x 104 (6.31 x 102 to 1.61 x 105) CD3+ cells/kg were given. DLI was given as early as 5 weeks in patients without evidence of GVHD. Inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions were assessed based on HLA and KIR genotyping of patients and their donors. Natural killer (NK) cells recovered relatively early, despite the presence of circulating MEDI-507. Split lineage MC was observed in each case, with a predominance of early donor myeloid chimerism and a lower percentage of donor T cell chimerism. Four of the 13 patients eventually lost their grafts despite DLI, including 2 who were not conditioned with fludarabine. Five patients spontaneously achieved full donor chimerism (FDC) and 4 converted to FDC following DLI. The spontaneous chimerism group had significantly fewer HLA mismatches in the HVG direction. Four out of the 5 had only one mismatch, whereas the patients who had graft loss or required DLI had at least two mismatches (p=0.007 by Fisher's exact test). This finding remained significant when the subgroup of patients not conditioned with fludarabine was excluded from the analysis (p=0.048). Spontaneous FDC was also associated with the presence of graft HLA-C group 1 (HLA-CAsn80) ligand for host NK cell inhibitory receptor KIR2DL2/3 (KIR-L match/compatible, 4 out of 4 evaluable patients) while this graft HLA ligand tended to be absent in the group that lost the graft or required DLI to achieve chimerism (KIR-L mismatch, 5 out of 8 patients) (p=0.08 by Fisher's exact test). There was no significant relationship between donor KIR and host HLA types in the GVH direction for predicting engraftment. While these observations are preliminary given the heterogeneity of the conditioning regimens and the small number of patients, the data suggest that the lesser degree of HLA mismatching and the presence of KIR ligand compatibility in the HVG direction may influence donor engraftment following nonmyeloablative TCD haploidentical HSCT and warrant further study.

Natural killer cell HLA‐C epitopes and killer cell immunoglobulin‐like receptors both influence outcome of mismatched unrelated donor bone marrow transplants

Matching of donor and recipient for the class I human leukocyte antigen-C (HLA-C)-encoded natural killer (NK) epitopes has been reported to influence stem-cell (SC) graft outcome, but a consistent picture has not yet emerged. We have analyzed transplant outcome in 104 unrelated SC grafts in relation to NK epitope (C1 and C2) matching and donor killer cell immunoglobulin-like receptor (KIR) genotype. NK epitope mismatching in the rejection direction was strongly associated with an increased probability of rejection subsequent to engraftment. The prevalence of grades III-IV acute graft-vs-host disease (GVHD) was significantly higher and occurred significantly earlier when there was NK epitope mismatching in the GVH direction. Higher transplant-related mortality and lower disease-free survival rates were associated with epitope mismatching regardless of the mismatch direction. A greater number of KIR receptors, both activating and inhibitory, in the donor protected against grades III-IV GVHD and improved survival.

NK-cell reconstitution after haploidentical hematopoietic stem-cell transplantations: immaturity of NK cells and inhibitory effect of NKG2A override GvL effect

AbstractNatural killer (NK) cell alloreactivity is reported to mediate strong GvL (graft versus leukemia) effect in patients after haploidentical stem-cell transplantation (SCT) for acute myeloid leukemia (AML). Because subsequent immune reconstitution remains a major concern, we studied NK-cell recovery in 10 patients with AML who received haplomismatched SC transplants, among whom no GvL effect was observed, despite the mismatched immunoglobulin-like receptor (KIR) ligand in the GvH direction for 8 of 10 patients. NK cells generated after SCT exhibited an immature phenotype: the cytotoxic CD3-CD56dim subset was small, expression of KIRs and NKp30 was reduced, while CD94/NKG2A expression was increased. This phenotype was associated to in vitro lower levels of cytotoxicity against a K562 cell line and against primary mismatched AML blasts than donor samples. This impaired lysis was correlated with CD94/NKG2A expression in NK cells. Blockading CD94/NKG2A restored lysis against the AML blasts, which all expressed HLA-E, the ligand for CD94/NKG2A. Our present study allows a better understanding of the NK-cell differentiation after SCT. These results revealed that the NK cells generated after haplomismatched SCT are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having potential impact for immune responsiveness and transplantation outcome.

Feasibility of HLA-haploidentical hematopoietic stem cell transplantation between noninherited maternal antigen (NIMA)–mismatched family members linked with long-term fetomaternal microchimerism

Based on the hypothesis that long-term fetomaternal microchimerism is associated with acquired immunologic hyporesponsiveness to noninherited maternal antigens (NIMAs) or inherited paternal antigens (IPAs), several groups have recently reported successful cases of non-T-cell-depleted hematopoietic stem cell transplantation (SCT) from HLA-haploidentical family members mismatched for NIMAs. In this study, we examined the outcomes of 35 patients with advanced hematologic malignancies who underwent HLA-2-antigen- or HLA-3-antigen-incompatible SCT from a microchimeric NIMA-mismatched donor. After standard-intensity or reduced-intensity preparative regimens, all patients had sustained hematopoietic recovery with tacrolimus-based graft-versus-host disease (GVHD) prophylaxis. Grade II/IV acute GVHD occurred in 19 (56%) of 34 evaluable patients, while extensive chronic GVHD developed in 13 (57%) of 23 patients who could be evaluated. Multivariate analysis demonstrated that NIMA mismatch in the GVH direction was associated with a lower risk of severe grade III-IV acute GVHD when compared with IPA mismatch (P = .03). Fifteen patients were alive and 14 of them were disease-free with a median follow-up of 20 (range, 8 to 37) months. These results indicate that T cell-replete SCT from an HLA-haploidentical NIMA-mismatched donor can offer durable remission with an acceptable risk of GVHD in selected patients with advanced hematologic malignancies who lack immediate access to a conventional stem cell source.

Comparison between Anti-Thymocyte Globulin and Alemtuzumab and the Possible Impact of KIR-Ligand Mismatch in Melphalan/Fludarabine Dose-Reduced Conditioning Followed by HLA-Matched and Mismatched Unrelated Stem Cell Transplantation in Patients with Multiple Myeloma.

We compared anti-thymocyte globulin (ATG-Fresenius median dose 60 mg/kg: n= 48) with alemtuzumab (Campath-1H 100mg: n=25) in 73 patients with multiple myeloma, who underwent dose-reduced conditioning with melphalan and fludarabine, followed by allogeneic stem cell transplantation from matched (n=63) or mismatched (n=10) unrelated donors. Patients of the ATG group had higher age (median 50 vs 47 years, p=0.05), more prior high-dose chemotherapies (p<0.001), while in the Campath group more bone marrow as stem cell source was used (p<0.001). No primary graft failure occurred in both groups. Patients receiving alemtuzumab had a significant faster engraftment of leukocyte (p=0.03) and of platelets (p=0.02) and a lower incidence of acute GvHD grade II-IV (24 vs 47%, p=0.05). However, after treatment with donor lymphocyte infusion due to persistent disease or mixed hematopoietic chimerism the difference of acute GvHD grade II-IV between alemtuzumab and ATG treated patients did not reach statistical significance (32 vs 47%, p=0.2). No difference in incidence of chronic GvHD was observed (25 vs 33%, p=0.6) More CMV seropositive patients in the alemtuzumab group experienced CMV reactivation (100% vs 47%, p=0.001). The cumulative incidence of treatment related mortality at 2 years for ATG and Campath was 29.3% (CI=17–50%) vs 28.5% (CI=15–54%), p=0.7. No significant difference could be observed in the estimated 2 years OS and PFS between ATG and Campath: 53% (CI:38–75) vs 45% (CI:28–73) and 29% (CI:16–54) and 36% (CI: 20–62), respectively. For PFS, in a multivariate analysis relapse to prior high-dose chemotherapy was the strongest negative factor: HR 2.9, p= 0.001. Including only those patients who did not experienced any relapse at time of allogeneic stem cell transplantation the Campath-group had a 2.5 fold higher risk of progression in comparison to the ATG group, but without reaching statistical significance (HR: 2.5, p=0.15). Ten out of 73 patients had KIR-ligand mismatch in GvH direction. While in patients without KIR-ligand mismatch the cumulative incidence of relapse at two years was 50%, none of the KIR-ligand mismatched patients relapsed so far (p=0.02). The immunosuppressive effect from Campath is stronger than ATG resulting in less acute GvHD, but requires more DLI procedures to control diseases and resulted in a trend to a lower PFS in chemosensitive patients. This preliminary data further implicated a major role of KIR-ligand mismatch transplantation in multiple myeloma

Reconstitution of NK Cells after Haploidentical Hematopoietic Transplants: Immaturity of NK Cells and Inhibitory Effect of NKG2A Override GvL Effect Expected by Alloreactive NK Cells.

The Perugia group reported favourable outcome in patients under haploidentical hematopoietic stem cell transplantation (SCT) for acute myeloid leukemia (AML) when donor and recipient were KIR ligand mismatched. In our study, we observed contradictory results in 11 adults (median age 25 years, range 15–38) who received a full haplotype mismatch SCT from a related donor at the Pitie-Salpetriere Hospital between October 1998 and December 2003 for high risk AML according to modalities of conditioning described by the Perugia group. Despite a KIR ligand mismatch in the GvH direction for 10 pairs of 11, majority of patients died (10/11) due to relapse for 7/11 and TRM for 3/11. Only 1 patient is alive in remission 8 months post SCT. No GvHD arose since the graft was positively selected for CD34+ cells using Clinimacs, resulting in an extensive T depletion. These observations incited us to perform in vitro studies to analyze NK cells reconstitution during the early period post haplo-SCT, as all the relapses occurred during the first 4 months following SCT. Peripheral blood was collected from 7 patients each month after SCT, and compared with the donors. T cells recovered very lately, in contrast to NK cells, which reached a normal absolute count rapidly. Similar phenotypic NK profile was observed in all patients. The CD56bright immunoregulatory NK cells subset was increased, representing a median of 55% of circulating NK cells at one month (M1), and 25 % at 3 months (M3) post SCT, as compared to 4% in the donors. Expression of the activating receptor NKp30 was reduced. The ratio of recipient/donor NKp30 on NK cells was 0.6 at M1 and 0.7 at M3. All inhibitory KIRs, especially, KIR2DL1 were down- regulated after SCT with a ratio, for KIR2DL1, at 0.23 at M1 and 0.37 at M3. To counterbalance the low expression of KIRs, the inhibitory receptor CD94/NKG2A was strikingly over expressed by NK cells generated after SCT (median of circulating NK cells expressing NKG2A: 97% at M1 and 90% at M3 versus 43% in the donors). This unusual phenotype persisted during all the study and evocated an immature state of NK cells. Immaturity of NK cells in vitro was associated with impaired functions. NK cells generated after SCT had a less efficient lysis of K562 cell line, and no cytotoxicity against the primary mismatched AML blasts, as compared to the donors. Expression of NKG2A by most NK cells post SCT was correlated with this impaired lysis. The ligand for NKG2A is HLA-E, which was also expressed by AML blasts as indicated by Western Blot analyzes. NK cells generated after SCT were cytotoxic against the HLA class I negative LCL 721-221cell line. Cytotoxicity was reduced against the LCL derivated 721-221 AEH cell line, transfected by HLA-E, and the lysis was restored after inhibition of NKG2A. Similar results were obtained with primary AML blasts; NK cytotoxicity against AML blasts was restored after blockade of NKG2A. We conclude that NK cells generated after haploidentical SCT exhibited an immature phenotype that persisted several months after SCT. This immaturity was correlated in vitro with an impaired cytotoxicity due to a dominant inhibitory role of NKG2A, and in vivo with the absence of GvL effect despite the mismatch KIR ligand in the GvH direction.

Are human platelet alloantigens (HPA) minor transplantation antigens in clinical bone marrow transplantation?

The role of human platelet alloantigens (HPA) in clinical bone marrow allotransplantation was investigated. The leading hypothesis was that HPA alloepitopes act as minor histocompatibility antigens and aggravate graft-versus-host disease (GVHD). To exclude the effect of MHC disparity, only HLA identical donor-recipient pairs were entered into the study. The influence of HPA compatibility on overall survival, occurrence of relapses and haematopoietic recovery was also investigated. A total of 223 patients who received a graft from an HLA-identical sibling, genotyped for HPA -1, -2, -3, -4 and -5, were observed over a post-transplant period of 24 months following the protocol recommended by EBMT. The data from patients having received grafts from HPA compatible donors were compared to data from patients having received grafts that were mismatched in HPA allotypes in the GVH direction. Analysis of the incidence of acute and chronic (GVHD), overall survival, relapse incidence, haematopoietic recovery and some other clinical parameters did not reveal any significant difference between the HPA-matched and -mismatched groups of patients, regardless of their age. Our results give no evidence that HPA-1, -2, -3 and -5 alloantigens should be considered minor transplantation antigens in clinical bone marrow transplantation.

Innate immunity against hematological malignancies.

Allogeneic hematopoietic transplantation relies on T-cell alloreactions for engraftment and the GvL effect. In HLA haplotype-mismatched transplants, extensive T-cell depletion of the graft is essential to prevent GvHD. This raises the question of whether mismatched transplants exert any GvL effect, and whether it will ever be possible to reduce the intensity of preparative regimens. Natural killer (NK) cells are negatively regulated by MHC Class I-specific inhibitory receptors. Mismatched transplants may therefore trigger NK-cell alloreactivity. The effects of NK-cell alloreactivity were evaluated in clinical transplantation and in murine transplant models. In clinical hematopoietic stem-cell transplants, HLA Class I disparities driving NK-cell alloreactions in the GvH direction eliminate AML relapse and graft rejection, while protecting patients from GvHD. In murine MHC mismatched transplant models, the pre-transplant infusion of donor-versus-recipient alloreactive NK cells conditioned the recipients to BMT, and reduced GvHD. NK-cell alloreactivity may thus provide a novel, powerful tool for enhancing the efficacy and safety of allogeneic hematopoietic transplantation.

Cellular therapy: exploiting NK cell alloreactivity in transplantation.

Allogeneic hematopoietic transplantation relies on T-cell alloreactions for engraftment and the graft-versus-leukemia (GVL) effect. In human leukocyte antigen (HLA) haplotype-mismatched transplants, extensive T-cell depletion of the graft is essential to prevent GVHD. This raises the question of whether mismatched transplants exert any GVL effect and whether it will ever be possible to reduce the intensity of preparative regimens. Because natural killer (NK) cells are negatively regulated by MHC class I-specific inhibitory receptors, mismatched transplants may trigger NK-cell alloreactivity. HLA class I disparities driving NK-cell alloreactions in the GVH direction mediate strong GVL effects, produce higher engraftment rates, and do not cause GVHD. In murine MHC-mismatched transplant models with no donor T-cell reactivity against the recipient, the pre-transplant infusion of donor-vs-recipient alloreactive NK cells conditioned the recipients to bone marrow transplantation without GVHD. NK-cell alloreactivity may be a unique therapeutic tool for tolerance induction and clearance of leukemia in hematopoietic transplantation.

Recognition of leukemic blasts by HLA-DPB1-specific cytotoxic T cell clones: a perspective for adjuvant immunotherapy post-bone marrow transplantation.

There is increasing evidence that the immune response plays a role in the prevention of leukemic relapses after allogeneic bone marrow transplantation (BMT). Producing this effect (referred to as the graft-versus-leukemia reaction or GVL) is a current goal of clinical transplantation. At present, all protocols rely on the injection of donor T cells with unknown specificities. In keeping with this approach, we recently proposed the use of a single allogeneic T cell clone transfected with the HSv-tk gene to target an HLA-DPB1 mismatch in the GVH direction. For this strategy to be successful, HLA-DP antigens must be expressed on leukemic cells, which should be recognised by the HLA-DP-specific T cell clone and subsequently destroyed. In the present study, differential expression of HLA-DR, -DQ and -DP was tested by fluorescence using monoclonal antibodies on a panel of 46 acute myeloid leukemias (AML), 28 acute lymphoblastic leukemias (ALL) and 31 chronic lymphocytic leukemias of B cell origin (B-CLL). The vast majority of leukemic cells expressed HLA-DP antigens although with considerable variability. HLA-DPB1 genotyped leukemic cells were used as target cells for an HLA-DPB1*0401-specific T cell clone. Specific recognition of leukemic blasts was demonstrated for 11 out of 11 B-CLL, 11 out of 19 AML and nine out of 16 ALL. These data show that most leukemic blasts are accessible to direct lysis by allogeneic HLA-DP-specific T cells.

Partially mismatched blood cell transplants for high-risk hematologic malignancy.

Eleven patients with high-risk hematologic malignancy received cryopreserved but otherwise unmanipulated blood cell transplants (BCT) from partially mismatched family members in whom progenitor cells had been mobilized by G-CSF. Donors were mismatched by up to one antigen in the GVH direction and up to three antigens in the rejection direction. Outcomes were compared with those of 22 patients receiving BCT from fully matched donors. Two mismatched patients died without engraftment on day 21 and 32. One had rejected bone marrow from the same donor, the other was mismatched by two antigens in the rejection direction and received the lowest dose of CD34+ cells. Median time to granulocyte engraftment was 21.5 (range 16-33) days for the mismatched group compared with 16 (11-28) days for the matched group (P = 0.01). No correlation was found between CD34+ cell dose and time to granulocyte or platelet recovery. In the mismatched and matched BCT groups respectively, the risk of grade II-IV acute graft-versus-host disease (GVHD) was 73% vs 28% (P = 0.001) and of chronic GVHD 100% vs 78% at 18 months (P = 0.01). The relationship of T cell dose to acute GVHD could only be evaluated in the matched group and no correlation was found. One of 11 mismatched patients and eight of 22 matched patients had relapse or persistent disease. Disease-free survival at 1 year was similar at 55% for mismatched and 50% for matched BCT. These results indicate that allogeneic BCT from partially mismatched family members is accompanied by a high incidence of GVHD but may result in comparable survival to BCT from fully matched donors.

Lack of correlation of MLC reactivity with acute graft-versus-host disease and mortality in unrelated donor bone marrow transplantation

Acute graft-versus-host disease (AGvHD) is a significant cause of morbidity and mortality in patients receiving a bone marrow transplant from an unrelated donor, and in an effort to reduce this problem, donors are selected for the least possible HLA incompatibility with the recipient. Selection criteria have included minimal incompatibility for the HLA-A, -B, and -DR loci and low reactivity in mixed lymphocyte culture (MLC); however, the value of MLC reactivity for prediction of development of AGvHD has been questioned. We therefore examined the correlation of MLC reactivity with AGvHD in recipients of unrelated bone marrow transplants. Reactivity in the GvH direction was assessed as relative response (RR) of donor lymphocytes to recipient stimulator lymphocytes. In 126 transplanted pairs with technically satisfactory MLC tests, the RR was divided into quartiles (0–1, 2–5, 6–16, and 17–117% RR). HLA-DRB1 incompatibilities were more frequent in the highest quartile ( p < 0.001); there were no significant differences among quartiles in donor or recipient age, diagnosis, or frequency of HLA-A or -B incompatibility. Incidence of AGvHD during the first 100 days post-transplant was assessed by Kaplan-Meier analysis. There was no significant difference in incidence of AGvHD among quartiles for the entire group of 126 pairs, for a subset with hematologie. malignancy, for a subset selected by a more stringent standard for “technically satisfactory” MLC, or for a subset matched for A, B, and DRB1. The MLC response of donor lymphocytes to recipient stimulator lymphocytes is thus not predictive of development of AGvHD in our patient population receiving unrelated donor bone marrow. Since there was no difference in mortality related to high and low MLC responses, our data also suggest that MLC results are not predictive of survival in this population. Human Immunology 49, 49–55 (1996)

HLA-DR, DQ, AND/OR DP GENOTYPIC MISMATCHES BETWEEN RECIPIENT-DONOR PAIRS IN UNRELATED BONE MARROW TRANSPLANTATION AND TRANSPLANT CLINICAL OUTCOME

Sixteen recipient-donor pairs who underwent unrelated BMT were analyzed for their HLA-class II identity by DNA-RFLP, in order to evaluate the importance of the genotypic HLA-DR, DQ, DP identity in the clinical outcome of unrelated bone marrow transplantation. From our study, a clear correlation between the HLA-DR, DQ, and DP genetic identity and acute GVHD (aGVHD) is not obvious since the number of studied cases is still limited. Nevertheless, it seems that the genetic identity influence the clinical outcome and patient survival. Six patients out of the ten who experienced severe aGVHD (greater than grade II) differed from their respective donors by HLA-DP mismatch in the GVH direction. Two patients rejected their grafts, and both presented HLA-DP incompatibilities in both GVH and HVG directions. Hence, HLA-DP may function as a transplantation antigen like the other HLA-class II molecules (DR, DQ) in unrelated BMT. Accordingly, we propose considering it in the pretransplantation histocompatibility testing. Nevertheless, further studies with larger numbers of cases should be done in order to confirm the role of HLA-DP. No correlation was observed between the mixed lymphocyte reaction (MLR) reactivity and the incidence of aGVHD. Accordingly, MLR response seems to be an incomplete indicator of GVHD, and a functional test is still to be found.